Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

Sensitivity of Ca2+ Transport of Mitochondria to Reactive Oxygen Species

The relationship between Ca²⁺ transport and energy transduction of myocardial mitochondria in the presence of reactive oxygen species was investigated. Following treatment with oxygen free radicals [superoxide(O ₂ ) or hydroxyl radical ()OH], lipid free radicals in myocardial mitochondrial membrane could be detected by using the method of EPR spin trap. Simultaneously there were obvious alterations in the free Ca²⁺ ([Ca²⁺]_m) in the mitochondrial matrix; the physical state of membrane lipid; the efficiency of oxidative phosphorylation (ADP/O); the value of the respiratory control ratio (RCR); and the membrane potential of the inner membrane of myocardial mitochondria. If the concentrations of reactive oxygen species were reduced by about 30%, the alterations in the physical state of the membrane lipid and energy transduction of myocardial mitochondria were not observed, but the changes in Ca²⁺ homeostasis remained. We conclude that Ca²⁺ transport by myocardial mitochondria is more sensitive to agents such as (O ₂ ) or OH, etc. than are oxidation phosphorylation and the respiratory chain.     

An insight in the mechanism of the aminoethylcysteine ketimine autoxidation

Summary Oxidation of aminoethylcysteine ketimine (AECK) is followed by the change of 296nm absorbance, by the O₂ consumption and by the HPLC analysis of the oxidation products. The oxidation is strongly inhibited by the addition of superoxide dismutase (SOD) but not by hydroxyl radical scavengers or catalase. Addition of EDTA or o-phenanthroline (OPT) favours the oxidation, probably by keeping contaminating metals in solution at the pH studied. Addition of Fe³⁺ ions strongly accelerates the oxidation in the presence of EDTA or OPT. AECK reacts stoichiometrically with OPT-Fe³⁺ complex producing the Fe²⁺ complex which is not reoxidised by bubbling O₂. HPLC analyses of the final oxidation products reacting with 2,4-dinitrophenylhydrazine (DNPH) confirm the AECK sulfoxide as the main product of the slow spontaneous oxidation. The detection of other oxidation products when the reaction is speeded up by the addition of the OPT-Fe³⁺ complex, suggests that the oxidation takes place essentially on the carbon portion of the AECK molecule in the side of the double bond. On the basis of the results presented here, a scheme of reactions is illustrated which starts with the transfer of one electron from AECK to a contaminating metal ion (possibly Fe³⁺) producing the radical AECK as the initiator of a self propagating reaction. The radical AECK reacting with O₂ starts a series of reactions accounting for most of the products detected.Abbreviations AECK S-aminoethyl-L-cysteine ketimine - AECK-SO aminoethylcysteine ketimine sulfoxide - CMCA S-carboxymethylcysteamine - DNPH 2,4-dinitrophenylhydrazine - OPT o-phenanthroline - DTPA diethylenetriaminepentaacetic acid - SOD superoxide dismutase     

Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (?)-epigallocatechin gallate (EGCG), (?)-epigallocatechin (EGC), and (?)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O₂^?) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O₂^? oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O₂^? oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO₄ by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (OH) and caused DNA damage. The OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H₂O₂ scavenger, inhibited the OH radical generation, indicating the involvement of H₂O₂ in the mechanism of Cr(VI)-induced OH generation. Catalase reduced OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating OH radicals are involved in the damage measured. The H₂O₂ formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO₄ impaired the generation of OH radical. The results obtained from this study show that reduction of insoluble PbCrO₄ by glutathione reductase/NADPH generates OH radicals. The mechanism of OH generation involves reduction of molecular oxygen to H₂O₂, which generates OH radicals through a Fenton-like reaction. The OH radicals generated by PbCrO₄ caused DNA strand breakage.     

Decay Kinetics of Tyrosine Radical (YZ •) in Chloride Anion-Depleted Photosystem 2 Studied by Time-Resolved EPR

The decay of tyrosine cation radical was found to be biphasic at 253 K. The fast phase corresponds to the Y_Z component while the slow phase corresponds to the tyrosine D radical (Y_D ) component. At 253 K, the t_1/2 value was 28.6 s for the fast phase and 190.7 s for the slow phase. The fast phase is attributed to the recombination of charges between Y_Z and Q_A ^–. The activation energy for the reaction of Y_Z with Q_A ^– between 253 and 293 K was 48 kJ mol^–1 in Cl^–-depleted photosystem 2 (PS2) membranes. Both the decay rate and the amplitude of the PAR -induced signal of Y_Z were affected by addition of chloride anion. Change in the decay rate and the amplitude of the PAR-induced signal of Y_Z was observed when other anions like Br^–, I^–, F, HCO₃ ^–, NO₃ ^–, PO₄ ^3– were substituted in the Cl^–-depleted PS2.     

An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

Green tea is rich in several polyphenols, such as (?)-epicatechin-3-gallate (ECG), (?)-epigallocatechin (EGC), and (?)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86?±?0.72?×?10⁴ M^?1 (K _ECG-HSA), 4.22?±?0.15?×?10⁴ M^?1 (K _{ECG-Cu(II)-HSA}), and 9.51?±?0.31?×?10⁴ M^?1 (K _{EGCG-Cu(II)-HSA}) at 293?K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.     

Evaluation of antioxidant activity of epigallocatechin gallate in biphasic model systems in vitro

The antioxidant activity of epigallocatechin gallate (EGCG) was studied in different in vitro model systems, which enabled evaluation of both chemical and physical factors involved in assessing the role of EGCG in oxidative reactions. EGCG suppressed the initiation rate and prolonged the lag phase duration of peroxyl radical-induced oxidation in a phospholipid liposome model to a greater extent (p < 0.01) compared to both Trolox and -tocopherol. Effectiveness of these antioxidants to prolong the peroxyl radical-induced lag phase was inversely related to lipophilic character. EGCG also protected against both peroxyl radical and hydroxyl radical-induced supercoiled DNA nicking. The rate constant describing EGCG reaction against hydroxyl radical was 4.22 ± 0.07 × 10¹⁰ M^–1·sec^–1, which was comparable to those of Trolox and -tocopherol, respectively. EGCG exhibited a synergistic effect with -tocopherol in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) radical, thus displaying a direct free radical scavenging capacity. In vitro Cu²⁺-induced-human LDL oxidation was accelerated in the presence of EGCG and attributed to the conversion of Cu²⁺ to Cu⁺. We conclude that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging. The same chemical-physical properties of EGCG also enabled prooxidant activity, only when in contact with unbound transition metal ions in a multiphasic system.     

H2O2 Sensors of Lungs and Blood Vessels and Their Role in the Antioxidant Defense of the Body

This paper considers the composition and function of sensory systems monitoring H₂O₂ level by the lung neuroepithelial cells and carotid bodies. These systems are localized in the plasma membrane of the corresponding cells and are composed of O ² -generating NADPH-oxidase and an H₂O₂-activated K⁺ channel. This complex structure of the H₂O₂ sensors is probably due to their function in antioxidant defense. By means of these sensors, an increase in the H₂O₂ level in lung or blood results in a decrease in lung ventilation and constriction of blood vessels. This action lowers the O₂ flux to the tissues and, hence, intracellular [O₂]. The [O₂] decrease, in turn, inhibits intracellular generation of reactive oxygen species. The possible roles of such systems under normal conditions (e.g., the effect of O ² in air) and in some pathologies (e.g., pneumonia) is discussed.     

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×10⁶ M⁻¹ and 1.66×10⁶ M⁻¹, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC₅₀ 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.     

Peroxynitrite-dependent modifications of tyrosine residues in hemoglobin. Formation of tyrosyl radical(s) and 3-nitrotyrosine

Summary. Although peroxynitrite is believed to be one of the most efficient tyrosine-nitrating species of biological relevance so far identified, its nitration efficiency is nevertheless limited. In fact, the nitrating species formed through peroxynitrite decay are caged radicals (OH/NO₂ or, in the presence of carbon dioxide, CO₃ ^–/NO₂) and the fraction that escapes from the solvent cage does not exceed 30–35%. One exception may be represented by metal-containing compounds that can enhance the formation of nitrotyrosine through a bimolecular reaction with peroxynitrite. Moreover, if the metal is also regenerated in the reaction, the compound is considered a nitration catalysts and the yield of tyrosine nitration enhanced several fold. Examples of peroxynitrite-dependent nitration catalysts are the Mn-superoxide dismutase, some cytochromes and several metalloporphyrins. On the contrary, it has been claimed that some hemoproteins are scavengers of peroxynitrite and play a role in limiting its biodamaging and bioregulatory activity. In this review, we discuss the case of hemoglobin, which is probably the major target of peroxynitrite in blood. This protein has been reported to protect intracellular and extracellular targets from peroxynitrite-mediated tyrosine nitration. This property is shared with myoglobin and cytochrome c. The possible mechanisms conferring to these proteins a peroxynitrite scavenging role are discussed.Present address: Laboratorio di Tossicologia Applicata ed Ecotossicologia, Istituto Superiore di Sanità, Rome, Italy.     

Quercetin inhibits hexose transport in a human diploid fibroblast

Summary The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK=7.65) is the inhibitory form of the molecule (K _I =10m). Quercetin, unlike phloretin, is rapidly degraded in 0.1n NaOH; the degradation products are weakly inhibitory to hexose transport.     

The effects of nitric oxide synthesis on the Na+,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilatör nitric oxide (NO). A major contributor to the increase in NO production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO^–) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO^– generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na⁺,K⁺-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na⁺,K⁺-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na⁺,K⁺-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na⁺,K⁺-ATPase activity and nTyr levels of LPS treated animals (r = –0.868, P = 0.001). Na⁺,K⁺-ATPase activity were also negatively correlated with iNOS activity (r = –0.877, P = 0.001) in inflamed kidney. These data suggest that NO and ONOO^– contribute to the development of oxidant injury. Furthermore, the source of NO may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO and ONOO^– formation inhibited Na⁺,K⁺-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na⁺,K⁺-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge. (Mol Cell Biochem 271: 107–112, 2005)     

The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition

Background

Much attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables.

Results

Our data indicate that human prostate cancer cell lines contain a small population of CD44⁺CD133⁺ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44⁺α2β1⁺CD133⁺ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSC's migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.

Conclusion

Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment.     

Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter

Exposure to ambient particulate matter has been reported to be associated with increased rates of lung cancer. Previously we showed that total suspended particulate matter (PM) induces oxidative DNA damage in epithelial lung cells. The aim of the present study was to further investigate the mechanism of PM-induced DNA damage, in which soluble iron-mediated hydroxyl radical (OH) formation is thought to play a crucial role. Using electron spin resonance (ESR) we showed that PM suspensions as well as their particle-free, water-soluble fractions can generate OH in the presence of hydrogen peroxide (H₂O₂), an effect which was abrogated by both deferoxamine and catalase. In addition, PM was also found to induce the OH-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the presence of H₂O₂ as assessed by dot-blot analysis of calf thymus DNA using an 8-OHdG antibody. In human alveolar epithelial cells (A549), both PM suspensions and the particle-free soluble fraction elicited formation of DNA strand breaks (comet-assay). Unlike the acellular DNA assays, in epithelial cells the DNA-damaging capacity of the particle suspensions appeared to be stronger than that of their corresponding particle-free filtrates. In conclusion, our findings demonstrate that the water-soluble fraction of PM elicits DNA damage via transition metal-dependent OH formation, implicating an important role of H₂O₂. Moreover, our data indicate that direct 'particle' effects contribute to the genotoxic hazard of ambient particulate matter in lung target cells.     

Cr(VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions

While Cr (VI)containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a timedependent manner. Nacetylcysteine (NAC), a general antioxidant, inhibited Cr (VI)induced tyrosine phosphorylation. Catalase, a scavenger of H₂O₂, sodium formate and aspirin, scavengers of hydroxyl radical (OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O₂ ^–), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of f Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H₂O₂ and OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).     

Biochemical studies on the lethal effects of solar and artificial ultraviolet radiation on Staphylococcus aureus

The effects of UV-B radiation generated in the laboratory and as a component of sunlight on the viability and particular biochemical activities of the bacterium Staphylococcus aureus have been examined. UV-B radiation progressively inhibits protein synthesis (assayed as ³H-alanine incorporation) and kills cells. Cell respiration, and RNA and DNA synthesis (³H-uridine and ³H-thymidine incorporation) were not greatly affected by UV-B irradiation. The OH and ¹O₂-free radical scavengers protected cells against killing and inhibition of protein synthesis by UV-B, suggesting that such radicals mediate the effects of UV-B on this organism. A similar protective effect using a ferric ion chelator suggests an important role for metallic ions in UV-B lethality.Abbreviations VIS, UV-A, UV-B, UV-C radiation in the bands 400–750 nm, 315–400 nm, 280–315 nm, 200–280 nm respectively - DBCO diazabicyclooctane - OFR oxygen free radical - OH, ¹O₂, O _inf2 ^sup- hydroxyl free radical, singlet oxygen, superoxide radical respectively     

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic ESTRFLP loci in the F₁(NA₆ í AU₆) population. A comprehensive set of ESTSSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA₆ genetic map contains 88 ESTRFLP and 71 ESTSSR loci with a total map length of 963 cM, while the AU₆ genetic map contains 67 ESTRFLP and 58 ESTSSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.An erratum to this article can be found at M.J. Faville and A.C. Vecchies contributed equally to this work.     

Differential role of hydrogen peroxide in UV-induced signal transduction

The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal Cl41 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H₂O₂ and O₂ ^–; fluorescence staining assay. Treatment of cells with UV radiation or H₂O₂ also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70^S6k in mouse epidermal JB6 cells. The scavenging of UV-generated H₂O₂ by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H₂O₂ inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70^S6k, while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an OH radical scavenger) or superoxide dismutase (O₂ ^– radical scavenger) did not inhibit any of these pathways. These results demonstrate that H₂O₂ generation is required for UV-induced phosphorylation of Akt and p70^S6k, and involved in activation of JNKs and p38 kinase, but not Erks.